Quality control and filtering results from cellranger

Sample info and environment setup

PRJNA732205

setwd("/media/jacopo/Elements/re_align/MM/PRJNA732205/SAMN19314102/SRR14629342/")
# Load the libraries (from Sarah script + biomart)
library(tidyverse) # packages for data wrangling, visualization etc
library(Seurat) # scRNA-Seq analysis package
library(clustree) # plot of clustering tree 
library(ggsignif) # Enrich your 'ggplots' with group-wise comparisons
library(clusterProfiler) #The package implements methods to analyze and visualize functional profiles of gene and gene clusters.
library(org.Hs.eg.db) # Human annotation package neede for clusterProfiler
library(ggrepel) # extra geoms for ggplo2
library(patchwork) #multiplots
library(reticulate)

Load and process cellranger data

Load and do the QC for the cellranger data

#list.files(".")
dat <- Read10X(data.dir ="./out/counts_filtered/")
dat <- CreateSeuratObject(dat) # Create the seurat object from the 10x data
kb.initial <- dat@assays[["RNA"]]@counts@Dim[[2]]
cat("Initial number of cells:", kb.initial, 
    "\nNumber of genes:",  dat@assays[["RNA"]]@counts@Dim[[1]])
## Initial number of cells: 2087 
## Number of genes: 36601

Quality Control

Empty cells were already filtered, check for % mt RNA and death markers:

# first calculate the mitochondrial percentage for each cell
dat$percent_mt <- PercentageFeatureSet(dat, pattern="^MT.")
# make violin plots
mt_rna = 20
max_counts = 30000



# Check some feature-feature relationships
# % mt RNA vs n Counts, n Features vs n Counts
# Check some feature-feature relationships
# % mt RNA vs n Counts, n Features vs n Counts
VlnPlot(dat, features = c("nCount_RNA", "nFeature_RNA", "percent_mt"))  + geom_hline(yintercept=mt_rna, linetype = "dotted")

plot1 <- FeatureScatter(dat, feature1 = "nCount_RNA", feature2 = "percent_mt")
plot1 <- plot1 + geom_hline(yintercept=mt_rna, linetype = "dotted")
plot2 <- FeatureScatter(dat, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
plot2 <- plot2 + geom_vline(xintercept = max_counts, linetype = "dotted")
plot1 

plot2

##  cells retained by mt RNA content ( 20 %): 1581 
##  percentage of retained cells: 75.75 %
## cells retained by counts ( 30000 ): 1579 
##  percentage of retained cells: 75.66 %

Check the distribution of the cells with low counts and control death markers:

min_counts = 350


hist(dat@meta.data$nCount_RNA, breaks = 100, xlab = "Counts")

hist(dat@meta.data$nCount_RNA, breaks = 1000, xlab = "Counts", xlim = c(0,5000))

hist(dat@meta.data$nCount_RNA, breaks = 10000, xlab = "Counts", xlim = c(0,1000))
abline(v=min_counts, col="red", lty = 3)

The evident peak of cells with < 200 counts could contain dying cells.

# Subset the dataset to focus only on those cells with low counts
dat.lowcount <- subset(dat, subset = nCount_RNA < min_counts)

# Get the mean of the counts for each gene and sort them decreasing
meanCounts <- rowMeans(GetAssayData(object = dat.lowcount, slot = 'counts'))
meanCounts <- sort(meanCounts, decreasing = T)

# A boxplot can help to observe the distribution of the means
#boxplot(meanCounts)

# Print the most highly expressed genes
head(meanCounts, 30)
##      IGLC1     MALAT1      IGHG3       IGKC      IGHGP     MT-ND2     MT-CO2 
## 45.7241379 24.2758621  9.8448276  8.8275862  6.3793103  3.1724138  1.8965517 
##     MT-CO3      IGHG1     MT-ND4     MT-ND1     MT-ND3     MT-CYB  MTRNR2L12 
##  1.8103448  1.7758621  1.5344828  1.3620690  1.3275862  1.3275862  1.2068966 
##    MT-ATP6     MT-CO1      IGHG2      RPS18        B2M      RPLP1       SSR4 
##  1.1379310  1.0172414  0.8965517  0.7758621  0.7586207  0.7586207  0.6896552 
##      RPL41   Z93241.1     MT-ND5      RPLP2      RPL13     RPL13A       TPT1 
##  0.6551724  0.6034483  0.5862069  0.5517241  0.5517241  0.5344828  0.4827586 
##      RPL10       MZB1 
##  0.4827586  0.4482759
## cells retained by counts ( 350 ): 1521 
##  percentage of retained cells: 72.88 %

dir.create("result")
saveRDS(dat, file = "./result/SAMN19314102_clean_QC.Rds")

Feature selection

#Normalize
dat <- NormalizeData(dat)
# Find the first 4000 variabe features
dat <- FindVariableFeatures(dat, selection.method = "vst", nfeatures = 4000)

Data scaling

Set mean expression to 0 and variance across 1 to avoid highly expressed genes drive the forwarding analyses. Since negative expression is meaningless, scaled data are useful only for UMAP and clustering

# scale data, the scaled data are saved in:
# dat[["RNA"]]@scale.data

all.genes <- rownames(dat)

dat <- ScaleData(dat, vars.to.regress = c("percent_mt","nCount_RNA"))

Dimensionality reduction

dat <- RunPCA(dat, features = VariableFeatures(object = dat), verbose = F, seed.use = 1)
print(dat[["pca"]], dims = 1:5, nfeatures = 5)
## PC_ 1 
## Positive:  IER2, NEAT1, TIMP1, RASD1, ID2 
## Negative:  RPS12, RPL13, RPS25, RPL19, SEC61G 
## PC_ 2 
## Positive:  PRDX1, HERPUD1, PSAT1, GLRX, PSMB1 
## Negative:  RPS19, RPLP2, RPS18, RPL13A, RPL34 
## PC_ 3 
## Positive:  IGKC, IGHGP, IGHG1, IGHG2, SSR4 
## Negative:  S100A4, NMI, RNF130, ZEB2, CAT 
## PC_ 4 
## Positive:  IGKC, ITM2C, EEF1A1, TMEM59, EEF1G 
## Negative:  JUN, HIST1H2BG, AL021155.5, Z93241.1, HSPB1 
## PC_ 5 
## Positive:  FTL, SRGN, TMSB4X, IER2, S100A10 
## Negative:  RPL13, IGHGP, RPS12, IGHG1, IGHG2

UMAP

UMAP is a graph-based method of clustering. The first step in this process is to construct a KNN graph based on the euclidean distance in PCA space:

dat <- FindNeighbors(dat, dims = 1:20)

The graph now can be used as input for the function runUMAP()

dat <- RunUMAP(dat, dims = 1:20, seed.use = 1)
DimPlot(dat, reduction = 'umap', seed = 1)

Final plots:

## QC metrics

## markers